PNITEBSITY  OF    CALIFORNIA   PUBLICATIONS 

COLLEGE  OF  AGRICULTURE 

AGRICULTURAL  EXPERIMENT  STATION 

BERKELEY,  CALIFORNIA 


BACTERIAL  DECOMPOSITION  OF 
OLIVES  DURING  PICKLING 

BY 

W.  V.   CRUESS  and   E.  H.  GUTHIER 


BULLETIN  No.  368 

July,  1923 


UNIVERSITY  OF  CALIFORNIA  PRESS 

BERKELEY,  CALIFORNIA 

1923 


Digitized  by  the  Internet  Archive 

in  2012  with  funding  from 

University  of  California,  Davis  Libraries 


http://www.archive.org/details/bacterialdecompo368crue 


BACTERIAL  DECOMPOSITION  OF 
OLIVES  DURING  PICKLING 

By  W.  V.  CRUESS  and  E.  H.  GUTHIER 


Picklers  of  ripe  olives,  since  the  beginning  of  the  industry  in  Cali- 
fornia, have  suffered  loss  of  fruit  during  pickling  because  of  bacterial 
decomposition.  The  financial  loss  during  the  1921  season  from  this 
cause  was  exceptionally  severe,  probably  in  excess  of  $75,000.  The 
loss  was  not  so  serious  during  the  1922  season,  largely  because  of  the 
introduction  of  control  measures  by  our  investigations. 

The  investigations*  reported  in  this  bulletin  were  made  during  the 
pickling  seasons  of  1921-22  and  1922-23. 

Nature  of  the  Decomposition. — In  the  usual  process  of  pickling 
ripe  olives,  the  fruit  is  first  treated  with  a  dilute  solution  of  lye 
(sodium  hydroxide)  long  enough  to  permit  the  lye  to  penetrate  the 
skin  and  a  short  distance  into  the  flesh.  The  olives  are  then  exposed 
to  the  air  in  the  vats  until  the  color  is  darkened  by  oxidation.  The 
first  exposure  usually  lasts  three  days  or  longer.  The  olives  are  then 
treated  with  several  applications  of  dilute  lye  and  are  exposed  to  the 
air  between  these  applications  for  periods  of  about  24  hours  each.  The 
final  lye  treatment  is  prolonged  sufficiently  to  destroy  all  bitterness. 
Water  is  then  placed  on  the  fruit  and  is  changed  several  times  daily 
until  the  lye  is  completely  washed  from  the  tissues.  Dilute  brine  is 
then  placed  on  the  olives  for  two  days  or  longer.  The  fruit  is  then 
sorted,  canned  and  sterilized  at  240°  F.  for  40  minutes. 

The  decomposition  occurs  more  frequently  during  the  washing  of 
the  fruit  following  the  final  lye  treatment  than  at  any  other  stage  of 
the  pickling  process. 

During  the  first  stages  of  the  disease  the  fruit  develops  a  character- 
istic sour  odor  and  flavor.  As  the  disease  progresses,  the  color  bleaches 
and  gas  is  formed  in  the  olives  and  in  the  brine.  "Floaters"  (fer- 
menting olives)  appear  on  the  surface  of  the  water  or  brine  in 
the  pickling  vats  and  bleaching  becomes  more  pronounced.  Finally 
putrefaction  occurs  and  the  olives  become  soft  and  inedible.  Gas 
pockets  develop  in  the  fruit  and  are  the  immediate  cause  of  floating. 
The  appearance  of  such  fruit  is  shown  in  figure  1. 


*  Grateful  acknowledgment  is  made  to  P.  H.  Waldruff,  L.  F.  Lingle  and  to 
many  packers  and  processors  for  their  cooperation  and  for  fruit  used  in  the  investi- 
gations. 


4  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

The  Organisms  Responsible  for  the  Disease. — Microscopical  exam- 
ination of  numerous  samples  of  fermented  olives  has  revealed  the  pres- 
ence of  large  numbers  of  bacteria.  The  predominant  organism  was 
found  to  be  a  very  short  rod,  occurring  singly  or  in  pairs.  A  long 
rod-like  bacterium  was  also  present  in  appreciable  numbers.  See 
figure  2  for  the  microscopical  appearance  of  these  organisms. 


Fig.  1. — Olives  showing  effects  of  bacterial  decomposition.  Some  of  these 
olives  are  cut  to  show  gas  pockets. 

It  was  found  possible  to  reproduce  the  disease  in  sterilized  olives 
by  inoculation  with  the  juice  from  affected  olives  or  by  addition  of 
such  olives  to  the  unaffected  fruit. 

Pure  cultures  of  the  organisms  from  fermenting  olives  were  pre- 
pared by  P.  H.  Waldruff  in  1922  and  by  Geo.  Ilenny  in  1923.  More 
than  twenty  different  types  were  obtained.  Waldruff  found  that  only 
two  of  his  pure  cultures  were  capable  of  causing  the  disease  and 
these  two  only  when  used  in  combination.     One  of  these  organisms 


Bulletin   368]  BACTERIAL   DECOMPOSITION  OF   OLIVES  5 

was  an  anaerobe  and  the  other  an  aerobe.  Henny  obtained  several 
pure  cultures  which  produced  gas.  The  senior  author  during  the 
1921  season  prepared  twelve  pure  cultures  of  aerobic  bacteria  but 
of  these  none  were  capable  of  causing  gas  formation  in  olives,  although 
several  caused  bleaching  of  the  color  and  formation  of  acid. 

It  is,  therefore,  probable  that  the  disease  is  caused  by  several  types 
rather  than  by  a  single  type  of  bacteria.  Some  varieties  cause  bleach- 
ing and  produce  acid  but  no  gas ;  others  produce  both  acid  and  gas. 


Fig.  2. — Organisms  from  olives  undergoing  decomposition  during  pickling. 
Magnified  about  1000  times. 

Sterilized  olives  were  inoculated  with  cultures  of  Bacillus  subtilis, 
Bacillus  coli  and  Bacillus  vulgaius,  because  it  was  suspected  that  one 
of  these  organisms  might  be  responsible  for  the  disease.  All  failed 
to  produce  gas,  although  growth  was  vigorous. 

The  resistance  to  heat  of  the  organisms  from  fermenting  olives 
and  to  sodium  hydroxide  is  discussed  later  in  this  publication. 

Sources  of  Infection. — In  order  to  determine  the  possible  sources 
of  the  bacteria,  pickled  olives  were  sterilized  in  3  per  cent  brine  in 
bottles  plugged  with  cotton.  To  different  samples  were  added:  (1) 
water  from  a  well  at  Lindsay;  (2)  olives  from  trees  at  Lindsay;  (3) 
unpickled  olives  from  holding  solution  (olives  grown  at  Lindsay)  ; 
(4)  piece  of  concrete  vat  in  which  olives  had  spoiled  during  pickling; 


b  UNIVERSITY    OF    CALIFORNIA- — EXPERIMENT    STATION 

(5)  piece  of  stave  of  barrel  in  which  unpickled  olives  had  been 
shipped;  and  (6)  piece  of  wooden  vat  in  which  olives  had  spoiled 
during  pickling. 

Sample  (1)  gave  a  negative  result;  samples  (2),  (3),  (4)  and  (6) 
caused  typical  fermentation  and  spoiling  when  tested  during  the  1921 
season;  samples  (5)  and  (6)  were  tested  during  the  1922  season, 
and,  although  putrefaction  occurred,  gas  formation  was  absent. 

Brine  and  water  from  fermenting  olives  were  added  to  sound  olives 
and  typical  fermentation,  floating  and  softening  ensued.  Unpickled 
olives  after  one  month 's  storage  in  brine  caused  the  disease  to  develop 
when  added  to  sterile  pickled  olives. 

These  results  indicate  that  the  bacteria  are  present  on  or  in  the 
olives  before  they  enter  the  factories.  It  is  also  evident  that  tanks  in 
which  spoiling  has  occurred  can  infect  sound  olives  placed  in  them. 
Olive  picklers  have  also  observed  that  olives  soften  and  ferment  more 
frequently  in  vats  in  which  such  spoiling  has  previously  occurred. 

Inoculation  of  sound  vats  of  olives  undoubtedly  frequently  occurs 
by  transfer  of  bacteria  from  one  vat  to  another  on  the  hands  of  the 
pickler  or  on  the  equipment  used  for  stirring  the  olives.  Picklers, 
therefore,  should  attempt  to  reduce  this  source  of  contamination  to  a 
minimum  by  sterilizing  dippers,  paddles  and  air  hose  in  a  disinfectant 
solution,  such  as  5  per  cent  formalin,  after  contact  with  fermenting 
olives. 

Relative  Susceptibility  of  Different  Varieties  of  Olives. — In  our 
experiments  it  was  observed  that  the  Sevillano,  Ascolano  and  Manza- 
nillo  olives  are  more  susceptible  than  the  Mission  to  bacterial  spoiling 
during  pickling;  the  relative  order  of  susceptibility  being  Sevillano, 
Ascolano,  Manzanillo  and  Mission.  It  was  found  very  difficult  to 
pickle  fresh  Sevillano  olives  in  the  laboratory  without  loss  of  much 
of  the  fruit  by  bacterial  decomposition. 

Little  difficulty  was  experienced  in  causing  fermentation  and  float- 
ing of  Sevillano,  Ascolano  and  Manzanillo  olives  experimentally ; 
Mission  olives  required  heavier  inoculations  and  much  longer  periods 
of  incubation  to  produce  the  disease.  After  the  appearance  of  fermen- 
tation, softening  progressed  more  rapidly  in  the  Sevillano,  Ascolano 
and  Manzanillo  varieties  than  in  the  Mission. 

Effect  of  Temperature  Baring  Pickling. — Losses  from  bacterial 
spoiling  of  Manzanillo  olives  have  been  heaviest  in  factories  in  which 
the  water  used  for  washing  is  above  70°  F.  In  factories  in  which  the 
vat  room  and  water  are  below  60°  F.  losses  in  most  instances  from 
this  source  have  been  small. 


Bulletin  368] 


BACTERIAL    DECOMPOSITION   OF    OLIVES 


In  order  to  obtain  more  accurate  information  on  the  relation  of 
temperature  to  bacterial  decomposition  the  four  most  important 
varieties  of  olives  were  pickled  to  the  point  of  application  of  wash 
water.  In  one  experiment,  samples  of  Mission,  Manzanillo  and  Asco- 
lano  olives  were  stored  at  temperatures  of  60°  F.,  80°  F.,  and  at  room 


Fig.  3. — Fermenting  olives  showing  floating  of  the  fruit  and  surface  growth. 

temperature  (50-70°  F.).  The  olives  were  inoculated.  Fermentation 
became  evident  at  80°  F.  in  less  than  36  hours  in  all  three  varieties ; 
in  the  Ascolanos  at  room  temperature  in  less  than  45  hours;  and  in 
the  Manzanillos  at  room  temperature  in  less  than  67  hours,  and  at 
60°  F.  in  144  hours.  The  Mission  olives  did  not  develop  fermentation, 
either  at  room  temperature  or  at  60°  F.,  during  the  period  (6  days) 
required  to  wash  the  lye  from  the  olive  tissues. 


8  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

In  another  experiment  Mission  and  Manzanillo  olives  were  washed 
at  60°  F.  and  at  78°  F.,  respectively.  Fermentation  of  the  Manza- 
nillos  at  78°  F.  became  evident  within  4  days,  and  only  after  16  days 
at  60°  F.  The  Missions  failed  to  develop  fermentation  at  60°  F.  and 
required  more  than  7  days  storage  at  78°  F.  to  develop  fermentation. 

Waldruff  was  unable  to  cause  fermentation  of  non-inoculated 
Manzanillo  olives  at  60°  F.,  whereas  fermentation  at  room  temperature 
occurred  in  less  than  one  week  in  olives  of  the  same  lot. 

Sevillano  olives  fermented  readily  at  60°  F.  In  vats  in  a  com- 
mercial factory,  Sevillanos  were  observed  to  be  fermenting*  at  45°  F. 

Other  experiments  confirmed  our  observation  that  Mission  and 
Manzanillo  olives  are  much  less  susceptible  to  bacterial  spoiling  at 
temperatures  below,  than  at  those  above,  60°  F. 

In  two  factories  which  have  experienced  heavy  losses  from  bacterial 
decomposition  of  olives  during  pickling,  the  temperature  of  the  water 
used  in  the  vat  room  was  from  72-76°  F.  In  three  other  factories  in 
which  bacterial  spoiling  during  pickling  has  been  rare  and  losses 
from  this  cause  have  been  almost  unknown,  the  temperature  of  the 
water  in  the  pickling  vats  was  less  than  50°  F. 

It  seems  evident  that  the  temperature  of  the  wash  water  is  a  very 
important,  if  not  the  most  important,  cause  for  the  development  of 
bacterial  decomposition  in  olives  during  pickling.  Those  who  con- 
template the  establishment  of  olive  pickling  factories  should  deter- 
mine the  temperature  of  the  water  available  for  use  in  pickling. 
Whether  artificial  cooling  of  the  water  would  be  economical  or  not 
has  not  been  determined  in  our  investigations.  It  is  possible  that 
other  means  of  preventing  bacterial  decomposition  discussed  later 
in  this  publication  would  be  more  feasible. 

Effect  of  Storing  Fruit  in  Brine  Before  Pickling. — It  was  observed 
that  of  two  factories  in  the  same  city  and  using  water  of  approxi- 
mately the  same  temperature  (70-75°  F.),  one  suffered  very  heavy 
losses  and  the  other  lost  relatively  little  fruit  by  bacterial  decompo- 
sition. Apparently,  the  principal  difference  in  the  pickling  processes 
in  the  two  factories  was  in  the  treatment  before  pickling.  In  the 
factory  in  which  losses  were  severe,  much  of  the  fruit  was  pickled 
direct  from  the  tree  or  after  a  short  storage  in  brine;  in  the  other 
factory  it  was  held  in  brine  for  several  weeks  to  several  months  before 
pickling. 

A  preliminary  experiment  made  in  the  1921-22  season,  indicated 
that  Manzanillo  olives  pickled  direct  from  the  tree  underwent  fermen- 
tation and  softening  readily,  whereas  those  pickled  after  six  months' 
storage  in  brine  failed  to  develop  these  symptoms,  even  when  heavily 


Bulletin   368]  BACTERIAL   DECOMPOSITION   OF   OLIVES  9 

inoculated.  During  the  1922-23  season  21  separate  lots  of  olives  of 
the  leading  varieties  were  pickled  both  before  and  after  storage  in 
brine  ("holding  solution").  Fifty-four  lots  of  olives  were  pickled 
in  the  21  experiments.  Brines  ranging  from  3  to  12  per  cent  salt 
were  used.     In  one  experiment  the  olives  were  stored  in  water  only. 

The  storage  period  ranged  from  26  to  120  days.  Fermentation 
occurred  in  the  unpickled  fruit  during  storage  in  brine,  but  appar- 
ently did  not  injure  its  texture.  Olives  placed  in  12  per  cent  salt 
solution  fermented  slowly  for  the  first  few  days  of  storage,  but  as  the 
brine  became  more  dilute  through  osmosis,  fermentation  became  more 
vigorous. 

The  brines  in  all  the  samples  decreased  in  salt  concentration  by 
osmosis.  For  this  reason  after  7  to  15  days'  storage,  salt  was  added 
to  increase  the  brines  to  the  original  concentrations  and  periodically 
thereafter  to  maintain  the  original  concentrations. 

The  various  samples  were  pickled  by  the  usual  lye  and  air  exposure 
process  described  on  page  one.  During  washing  to  remove  the  lye  the 
olives  were  inoculated  with  crushed  fermenting  olives  to  induce  fer- 
mentation and  softening  in  susceptible  olives. 

All  experimental  lots  of  olives  not  previously  stored  in  brine 
developed  the  disease  very  quickly  and  in  a  vigorous  form.  Those 
stored  26  days  or  longer  in  brine  either  did  not  develop  the  disease, 
or  developed  it  only  after  prolonged  standing,  e.g.  15  to  18  days.  The 
results  were  very  striking  and  conclusive.  Olives  of  several  lots  which 
failed  to  ferment  were  sterilized  in  a  3  per  cent  salt  solution  contain- 
ing 1  per  cent  of  sugar.  After  inoculation,  fermentation  occurred 
within  3  days  at  room  temperature,  indicating  that  sugar  favors 
fermentation. 

Olives  stored  in  brines  of  10  to  12  per  cent  salt  were  firmer  when 
pickled  than  those  stored  in  brines  of  3  to  6  per  cent,  or  pickled  with- 
out storage  in  brine. 

It  is  probable  that  storage  in  brine  before  pickling  permits  decom- 
position of  sugar  and  other  fermentable  substances  so  that  when  the 
fruit  is  pickled  later  there  are  none  of  these  materials  to  support  fer- 
mentation. Fermentation  of  the  unpickled  fruit  apparently  did  not 
injure  it. 

CONTROL    MEASURES 

Several  methods  of  preventing  and  checking  the  disease  during 
the  pickling  process  were  studied,  both  in  the  laboratory  and  under 
factory  conditions. 

Hastening  the  Washing  Process. — Fermentation  and  softening 
most  frequently  occur  during  the  washing  of  the  olives  in  water  to 


10 


UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 


remove  the  lye.  Therefore,  our  efforts  to  shorten  the  pickling  process 
were  applied  principally  to  this  step  in  the  work. 

Olives  of  the  same  variety  were  pickled  to  the  point  of  adding 
the  first  wash  water.  They  were  divided  into  five  lots  and  water 
applied  at  140°  F.,  97°  F.,  77°  F.,  67°  F.,  and  37°  F.}  respectively. 
The  water  was  changed  twice  daily  on  all  lots  except  that  at  140°  F., 
on  which  it  was  changed  three  times  daily.  All  lots  except  that  at 
140°  F.  were  maintained  constantly  at  the  respective  temperatures. 
Water  at  140°  F.  was  placed  on  this  lot  of  olives,  but  it  was  allowed 
to  cool  naturally  to  75-100°  F.  between  additions  of  water. 

The  times  necessary  at  each  temperature  to  completely  remove  the 
lye  from  the  flesh  of  the  fruit  are  shown  graphically  in  figure  4. 


>40 
'50 

too 

"O 

/? 

CLfl 

T/OA 

'  or 

TEt 

IPC/ 

?/m 

//TC 

TV 

<oo 

77/ 

1C  f\ 

EQL 

'/R£ 

D   TC 

'  w/ 

'v5// 

tyt 

90 

Ff\ 

'0/7 

OLI\ 

'€ 

T/SZ 

U£ 

m 

r, 

*) 

^ 

uj 

m 

k 

'iO 

^ 

40 

IS 

3C 

77M£ 

/ft 

HOU 

^5 

"V 

D 

-<70 

A 

00 

>S 

oo 

j 

oo 

"K>0 

Fig.  4. — Effect  of  temperature  of  wash  water  on  time  required  to  remove  lye 
from  olive  tissue. 


At  97°,  77°,  and  67°  F.  fermentation  occurred  but  not  at  37°  F. 
nor  at  140°  F.  It  is  probable  that  a  temperature  of  140°  F.  retarded 
bacterial  growth,  in  addition  to  greatly  hastening  the  washing  process. 

One  factory  has  used  wash  water  at  about  140°  F.  with  success. 

Pasteurization. — A  large  number  of  experiments  were  made  during 
the  past  two  seasons  to  determine  the  death  temperature  of  the  organ- 
isms responsible  for  the  disease.  Two  experiments  will  be  cited  to 
illustrate  the  methods  used. 

In  Experiment  390,  fermenting  pickled  olives  were  crushed  in  a 
sterile  mortar  and  pressed  through  sterile  cheesecloth.  The  juice  was 
mixed  with  sterile  water.     Samples  were  heated  to  175°   F.  for  30 


Bulletin  368] 


BACTERIAL    DECOMPOSITION   OF    OLIVES 


11 


and  45  minutes,  respectively.  Surface  cultures  of  the  heated  and  of 
the  untreated  samples  were  made  on  nutrient-dextrose  agar  agar  to 
determine  the  total  number  of  aerobic  bacteria  present,  and  deep 
cultures  in  dextrose  agar  agar  to  determine  whether  or  not  gas  form- 
ing bacteria  was  present. 


Fig.  5. — Effect  of  pasteurization  on  organisms  from  fermenting  olives.  No.  1, 
heated  to  175°  F.  for  30  minutes;  No.  2,  not  heated.  Note  breaking  of  the  agar 
agar  jelly  in  No.  2  by  gas. 


The  untreated  sample  contained  13,000,000  aerobic  organisms  per 
c.c. ;  those  heated  to  175°  F.  for  30  and  45  minutes  contained  no 
aerobic  bacteria. 

The  deep  culture  of  the  untreated  sample  quickly  developed  gas; 
that  from  the  sample  heated  to  175°  F.  for  30  minutes  developed  a 


12  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

single  colony  from  1  c.c.  of  sample  but  no  gas,  and  that  heated  to 
175°  F.  for  45  minutes  developed  neither  colonies  nor  gas.  Figure  5 
illustrates  the  appearance  of  the  deep  cultures  from  an  untreated 
check  (No.  2)  and  from  the  sample  heated  to  175°  F.  for  30  minutes 
(No.  1).  Note  breaking  of  the  agar  agar  in  sample  No.  2,  positive 
evidence  of  the  presence  of  gas  producing  organisms. 

Fermenting  pickled  Manzanillo  olives  were  divided  into  ten  lots 
which  were  then  heated  to  212°  F.  for  15  minutes;  190°  F.  for  15 
minutes;  170°  F.  for  15,  30,  45,  and  60  minutes;  and  to  150°  F.  for 
15,  30,  45  and  60  minutes,  respectively.  The  samples  were  then  stored 
in  sterile  bottles  plugged  with  cotton  for  about  30  days.  Samples 
heated  to  212°  F.  for  15  minutes;  190°  F.  for  15  minutes;  170°  F.  for 
60  minutes,  and  150°  F.  for  60  minutes  did  not  develop  fermentation. 
All  other  samples  fermented  in  less  than  seven  days.  Other  experi- 
ments confirmed  these  results. 

A  longer  period  of  heating  was  found  necessary  with  the  whole 
olives  than  with  the  juice,  probably  because  considerable  time  was 
required  for  the  flesh  near  the  pits  of  the  olives  to  reach  the  tempera- 
ture of  the  surrounding  heated  liquid.  A  temperature  of  190°  F. 
for  a  period  of  15  minutes  would  appear  to  be  the  most  economical 
for  pasteurizing. 

In  commercial  factories,  pasteurizing  fermenting  olives  at  175°  F. 
for  30  minutes  was  found  effective  in  checking  fermentation  long 
enough  to  remove  the  lye  from  the  olives  by  washing  with  water  in 
the  usual  manner.  The  method,  therefore,  has  proved  to  be  applicable 
to  factory  practice. 

Fermenting  olives  exhibited  some  softening  when  pasteurized  in 
the  vats  by  direct  steam,  because  of  mechanical  bruising  by  the  steam 
jet.  Some  method  of  heating  the  water  or  brine  outside  the  vat  should 
be  employed. 

Olives  which  contained  large  gas  pockets  and  were  floating  usually 
softened  when  heated.  Therefore,  pasteurization  should  be  applied 
before  fermentation  has  progressed  to  the  point  of  causing  floating 
of  the  olives.  Pasteurized  olives  should  also  be  sorted  in  some  cases 
to  remove  softened  fruit.  The  softened  fruit  can  usually  be  identified 
by  its  collapsed  or  wrinkled  appearance  after  heating. 

Control  by  Lye  Treatment. — In  the  laboratory,  fermenting  pickled 
olives  were  treated  for  various  lengths  of  time  with  lye  solutions  vary- 
ing from  %  Per  cent  to  IV2  per  cent  sodium  hydroxide  (lye).  The 
y4  per  cent  and  %  Per  cent  solutions  failed  to  prevent  subsequent 
fermentation  during  washing;   %   per   cent   and   stronger   solutions 


BULLETIN    368]  BACTERIAL    DECOMPOSITION   OF    OLIVES  13 

applied  for  about  15  hours  prevented  subsequent  fermentation,  but 
application  for  3  to  8  hours  in  most  eases  failed  to  check  the  disease. 

Treatment  of  actively  fermenting  olives  in  the  vats  in  factory  scale 
experiments  with  %  to  1  per  cent  lye  solutions  to  the  pits  (about 
3  to  5  hours  application)  failed  to  check  fermentation  long  enough 
to  permit  washing  out  of  the  lye  in  the  usual  manner.  Similar  treat- 
ment of  olives  in  the  first  stages  of  fermentation,  however,  has  proved 
effective  in  checking  the  disease  in  many  instances. 

In  one  experiment  fermenting  olives  were  crushed  and  pressed 
with  sterile  equipment  and  the  juice  was  mixed  with  sterile  water. 
Samples  of  the  liquid  were  treated  for  35  minutes  and  for  3  hours 
with  14,  y2,  and  %  per  cent  lye  solutions.  Counts  of  surviving  bac- 
teria were  made  by  planting  on  nutrient  agar  agar  and  qualitative 
tests  for  the  presence  of  gas  forming  bacteria  were  made  in  dextrose 
agar  tubes.  The  %,  %,  and  %  per  cent  lye  solutions  for  3  hours 
destroyed  the  gas  forming  bacteria,  but  a  few  non-gas  forming  organ- 
isms survived. 

In  another  experiment  fermenting  olives  of  the  same  lot  were 
treated  at  room  temperature  for  5  hours  and  for  23  hours  with  lye 
solutions  of  V2?  %,  and  1  per  cent.  No  gas  forming  bacteria  were 
recovered  from  any  of  the  samples  after  23  hours  treatment  and  after 
5  hours  treatment  with  1  per  cent  lye  solution.  The  numbers  of 
aerobic  bacteria  in  the  olives  before  and  after  treatment  are  shown 
in  the  following  table. 

TABLE  1 

Effect  of  Lye  Concentration  and  Length  of  Lye  Treatment  on 
Aerobic  Bacteria  in  Fermenting  Olives 

Aerobic  bacteria  per  c.c. 

a 

After  5  hours  After  23  hours 

Sample  treatment  treatment 

Untreated  425,000,000  425,000,000* 

y2  per  cent  lye 320,000  More  than  40,000 

%  per  cent  lye 960,000  80 

1  per  cent  lye 0  0 

*  Assumed  to  be  the  same  as  on  the  previous  day. 

In  several  other  experiments  these  results  were  confirmed,  but  in 
one  experiment  treatment  of  the  olives  with  a  %  per  cent  solution 
for  24  hours  failed  to  prevent  fermentation  of  the  olives  during 
subsequent  washing. 

It  seems  evident  from  our  experiments  that  a  %  per  cent  lye 
solution  applied  for  20  to  24  hours,  or  a  1  per  cent  lye  solution  applied 


14  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

for  a  shorter  time,  will  "cure"  mild  cases  of  the  disease,  but  that 
this  treatment  is  not  adequate  for  severe  cases. 

Control  by  Pasteurization  and  Subsequent  Lye  Treatment. — 
Bacterial  growth  in  olives  during  washing  causes  bleaching ;  probably 
by  the  production  of  acid  and  reduction  of  the  color  (the  reverse  of 
oxidation).  It  was  found  that  most  of  the  lost  color  returned  when 
the  olives  were  given  a  light  lye  treatment  and  exposed  to  the  air 
for  24  hours. 

Because  a  light  lye  treatment  used  alone  failed  to  permanently 
check  fermentation,  it  was  found  advisable  first  to  pasteurize  the 
fermenting  olives,  then  to  apply  a  dilute  lye  solution  (%  per  cent) 
to  the  pit  (2  to  4  hours  application),  expose  them  to  the  air  for  24 
hours,  and  wash  free  from  lye  in  the  usual  manner. 

SUMMARY 

1.  Bacterial  decomposition  (fermentation,  floating,  and  soften- 
ing) of  olives  has  in  the  past  caused  serious  loss  to  olive  packers. 

2.  It  is  caused  by  gas-forming  and  other  bacteria  which  are  present 
on  the  surface  or  in  the  fresh  fruit,  the  vats  and  other  equipment 
before  pickling. 

3.  The  presence  of  fermentable  material  (sugars  or  similar  com- 
pounds) in  the  fruit  is  essential  for  the  typical  development  of  the 
disease. 

4.  Storage  in  brine  for  four  weeks  or  longer  permits  fermentation 
of  this  material  before  pickling  without  injury  to  the  fruit  and 
thereby  renders  the  fruit  resistant  to  fermentation  during  pickling. 

5.  Sevillano,  Ascolano,  and  Manzanillo  olives  are  much  more 
susceptible  than  the  Mission  to  bacterial  decomposition  during 
pickling. 

6.  The  use  of  wash  water  above  60°  F.  greatly  increases  the 
susceptibility  of  the  olives  to  fermentation. 

7.  Pasteurization  at  212°  F.  or  190°  F.  for  15  minutes  or  at 
175-170°  F.  for  60  minutes  checks  fermentation  long  enough  to  permit 
washing  the  lye  from  the  tissues  of  the  olives.  Pasteurization  at 
175°  F.  for  30  minutes  has  proved  efficacious  in  control  of  the  disease 
under  factory  conditions. 

8.  Application  of  %-l  per  cent  lye  for  23  to  24  hours  checks  the 
disease  in  mild  cases  long  enough  to  permit  completion  of  the  pickling 
process,  but  is  not  sufficient  for  cure  of  advanced  cases. 


Bulletin  368]  BACTERIAL  DECOMPOSITION  OF  OLIVES  15 

9.  Application  of  dilute  lye  and  exposure  to  air  after  pasteuriza- 
tion improves  the  color  of  olives  bleached  by  bacterial  action. 

10.  Application  of  wash  water  at  140°  F.  reduces  the  time  neces- 
sary for  washing  lye  from  the  olive  tissues  to  about  two  days.  Six 
to  eight  days  is  normally  required  at  50-60°  F. 

RECOMMENDATIONS 

1.  Manzanillo,  Sevillano  and  Ascolano  olives  should  be  stored  in 
brine  for  at  least  25  days  before  pickling.  A  brine  of  about  6-7 
per  cent  salt  (24-28°  salometer)  for  the  first  week  with  an  increase 
to  10  per  cent  (40°  salometer)  for  the  remainder  of  the  storage  period 
is  recommended. 

2.  In  establishing  a  new  olive  pickling  factory,  the  prospective 
builder  should  make  certain  that  water  not  above  60°  F.  is  available. 

3.  In  factories  where  the  wash  water  is  cold  (40-50°  F.)  it  is 
recommended  that  fermentation  be  arrested  by  lye  treatment. 

4.  In  factories  using  warm  water  (60°  F.  or  above)  olives  fer- 
menting during  pickling  should  be  pasteurized  at  190°  F.  for  15 
minutes;  cooled  to  room  temperature;  treated  with  dilute  lye  (%— % 
per  cent)  to  the  pit ;  exposed  24  hours  to  the  air  to  darken,  and  washed 
free  from  lye  as  rapidly  as  possible. 

5.  In  factories  in  which  bacterial  decomposition  has  become 
thoroughly  established,  the  vats  should  be  sterilized  with  boiling  water 
and  great  care  exercised  in  order  to  avoid  transfer  of  bacteria  from 
infected  to  sound  vats  of  olives  by  the  hands,  stirring  paddles,  or  other 
implements. 

6.  In  such  factories  the  washing  process  should  be  hastened  as 
much  as  possible,  one  effective  means  being  the  application  of  hot 
wash  water  (140°  F.)  to  hasten  leaching  of  lye  from  the  olives.  The 
fruit  should  be  stored  no  longer  than  necessary  in  dilute  brine  after 
pickling,  and  should  be  canned  as  soon  as  possible  when  free  from  lye. 
Storage  for  two  days  in  brine  before  canning  should  be  the  maximum 
in  factories  in  which  bacterial  decomposition  is  a  serious  problem. 


